adsl  (Proteintech)

Journal: Biofactors (Oxford, England)

Article Title: Diverse Inhibitors of De Novo Purine Synthesis Promote AICAR ‐Induced AMPK Activation and Glucose Uptake in L6 Myotubes

doi: 10.1002/biof.70037

Figure Lengend Snippet: Assessment of sensitivity of L6 cells to inhibitors of purine metabolism. A: Purine metabolism and AMPK. The purine precursor ZMP is an AMPK activator. Methotrexate was shown to promote fatty acid oxidation (FAO) and glucose uptake via activation of AMPK in skeletal muscle tissue or cells [ , ]. Intermediates : 5,10‐CH 2 ‐THF, N 5 ,N 10 ‐methylene THF; 10‐CHO‐THF, N 10 ‐Formyl‐THF; AMP, adenosine monophosphate; DHF, dihydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FGAR, formylglycinamide ribonucleotide; GAR, glycinamide ribonucleotide; GMP, guanosine monophosphate; Hx, hypoxanthine; IMP, inosine monophosphate; PRA, phosphoribosylamine; PRPP, 5‐phosphoribosyl‐1‐pyrophosphate; SAICAR, N‐succinyl‐5‐aminoimidazole‐4‐carboxamide ribonucleotide; THF, tetrahydrofolate; Xan, xanthine; ZMP, 5‐aminoimidazole‐4‐carboxamide ribonucleotide. Enzymes : ACC, acetyl‐coenzyme A carboxylase; ADSL, adenylosuccinate lyase; ADSS, adenylosuccinate synthetase; AMPK, AMP‐activated protein kinase; ATIC, 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase; DHFR, dihydrofolate reductase; GART, glycinamide ribonucleotide formyltransferase; GMPS, GMP synthetase; GPAT, glutamine phosphoribosylpyrophosphate amidotransferase; IMPDH, IMP dehydrogenase; TS, thymidylate synthetase; XDH, xanthine oxidase. Inhibitors : ALA, alanosine; ALO, allopurinol; FAO, fatty acid oxidation; MMF, mycophenolate mofetil; MP, mercaptopurine; MTX, methotrexate; TMP, trimethoprim; TMX, trimetrexate. “P” on AMPK and ACC indicates phosphorylation. (B, C) L6 Myotubes express enzymes of nucleotide and folate metabolism targeted by MTX, ALA, MMF, MP, TMX, and ALO. L6 cells were grown for 2 days in MEMα with nucleosides and 10% serum and then differentiated for 7 days in MEMα with nucleosides and 2% serum and for an additional day in MEMα without nucleosides and serum. Cells were then analyzed for expression of Gart , Atic , Adss1 , Adss2 , Adsl , Impdh1 , Impdh2 , Tyms , Dhfr , and Xdh genes (B). Expression of target genes was normalized to expression of Actin beta gene ( Actb ). Graphs show means with SD ( n = 2). Tyms : Thymidylate synthetase. In addition, cells were analyzed for protein expression of GART, ATIC, ADSS, IMPDH2, DHFR, and XDH on day 2 (myoblasts; MB), day 9 (myotubes after 7 days in MEMα with nucleosides and 2% serum; MT+) and day 10 (myotubes after 7 days in MEMα with nucleosides and 2% serum and 1 day in MEMα without nucleosides and serum; MT‐) of culture (C). Numbers next to blots indicate position and molecular weight (in kDa) of molecular weight markers. (D–J) Effect of MMF, ALA, MP, TMP, sulfamethoxazole (SMX), TMX and MTX on proliferation of L6 myoblasts in absence or presence of nucleosides. L6 myoblasts were grown in absence of nucleosides for 24 h and then treated with MMF (0.1–10 μM) (D), ALA (0.1–10 μM) (E), MP (1–100 μM) (F), TMP (1–100 μM) (G), SMX (10–1000 μM) (H), TMX (0.1–10 μM) (I), MTX (0.1–10 μM) (J) or vehicle (control, C) in absence or in presence of nucleosides for 48 h. Cell cultures before and after the treatment were analyzed for DNA content with Hoechst assay. Hoechst fluorescence (Hoechst FL) after the treatment was expressed relative to Hoechst fluorescence before the treatment (0 h). Graphs show means with SD ( n = 4–8). * p < 0.05 versus respective (without or with nucleosides) control, two‐way ANOVA with Dunnett's test.

Article Snippet: Quantitative real‐time polymerase chain reaction (qPCR) was performed with QuantStudio 3 Real‐Time PCR System (Thermo Fisher Scientific) using TaqMan Universal PCR Master Mix II and TaqMan gene expression assays for Gart (Rn01477298_m1), Atic (Rn00578818_m1), Adss1 (Rn01430183_m1), Adss2 (Rn02103847_s1), Adsl (Rn01768239_m1), Impdh1 (Rn01455843_g1), Impdh2 (Rn01640111_g1), Tyms (Rn01418709_m1), Dhfr (Rn04342282_g1), Xdh (Rn00567654_m1) and Actb (4352931).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Molecular Weight, Control, Fluorescence

Journal: Advanced Science

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma

doi: 10.1002/advs.202416809

Figure Lengend Snippet: LEDGF reads H3R17me2a regulating key enzymes in the de novo synthesis pathway. A) Heat maps and averaged CUT&Tag signals of H3R17me2a and LEDGF across ±5 kb from the transcription start site (TSS) in A498 cells. B) Distribution of H3R17me2a and LEDGF enrichment peaks on the genome. C) Motif analysis of high frequency enrichment of H3R17me2a and LEDGF shows that there is a high degree of co‐enrichment in the genome. D) There are specific enrichment peaks at the TSS of PPAT , PAICS , GART , ADSL , and ADSS2 in indicated groups, suggesting a potential transcriptional regulatory axis in A498 cells. E–H) The specific enrichment of H3R17me2a at the TSS of PPAT , PAICS (E), GART (F), ADSL (G), and ADSS2 (H) was verified by ChIP‐qPCR assay. I–L) The specific enrichment of LEDGF at the TSS of PPAT , PAICS (I), GART (J), ADSL (K), and ADSS2 (L) was verified by ChIP‐qPCR assay. M,N) QRT‐PCR was used to demonstrate that decrease of LEDGF or H3R17me2a can reduce mRNA expression of key enzymes in the de novo synthesis pathway. (N) Western blot was performed to detect that decrease of LEDGF or H3R17me2a can significantly reduce the protein expression of key enzymes of de novo synthesis, except ADSS2. O–R) Metabolomics results showed that reduction of H3R17me2a level or LEDGF significantly decreases IMP (O) and GMP (P) levels in A498 cells. While the AMP in A498 cells remain relatively stable (Q), there is a significant reduction in dAMP (R) level. Data are shown as mean ± SD. ** p < 0.01, *** p < 0.001. ns means no significance.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401‐1‐AP), PAICS (Proteintech, #12967‐1‐AP), GART (Proteintech, #13659‐1‐AP), ADSL (Proteintech, #15264‐1‐AP), ADSS2 (Proteintech, #16373‐1‐AP), Histone H3 (Proteintech, #17168‐1‐AP), Alpha Actin (Proteintech, #23660‐1‐AP), Flag (Abmart, #M20008), Goat Anti‐Rabbit IgG (H + L) (Proteintech,#SA00001‐2), and Goat Anti‐Mouse IgG (H + L) (Proteintech, #SA00001‐1).

Techniques: ChIP-qPCR, Quantitative RT-PCR, Expressing, Western Blot

Journal: Advanced Science

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Deficiency of LEDGF protects NKG mice against xenograft proliferation. A) Schematic diagram of subcutaneous tumor model in NKG mice in indicated treatment groups. All surviving mice were euthanized 8 weeks after tumor cell inoculation. B) Knock out of LEDGF effectively reduced the proliferation of xenografts in NKG mice. (n = 5) C) There was no significant difference in body weight between the two groups throughout the experiment. D–F) Elimination of LEDGF effectively reduced the volume (D‐E) and weight (F) of NKG mice xenografts. G) QRT‐PCR was used to demonstrate that decrease of LEDGF can reduce mRNA expression of PPAT, PAICS, GART, ADSL, and ADSS2 in xenograft tumors. H) The proliferation ability of xenografts in LEDGF‐KO group was significantly reduced. The expression levels of PPAT, PAICS, GART, and ADSL were significantly decreased, while ADSS2 expression was almost unchanged. Scale bar = 100 µm. I) A schematic model illustrating that LEDGF interacts with CARM1‐mediated H3R17me2a to promote ccRCC progression. Data are shown as mean ± SD. *** p < 0.001. ns means no significance.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401‐1‐AP), PAICS (Proteintech, #12967‐1‐AP), GART (Proteintech, #13659‐1‐AP), ADSL (Proteintech, #15264‐1‐AP), ADSS2 (Proteintech, #16373‐1‐AP), Histone H3 (Proteintech, #17168‐1‐AP), Alpha Actin (Proteintech, #23660‐1‐AP), Flag (Abmart, #M20008), Goat Anti‐Rabbit IgG (H + L) (Proteintech,#SA00001‐2), and Goat Anti‐Mouse IgG (H + L) (Proteintech, #SA00001‐1).

Techniques: Knock-Out, Quantitative RT-PCR, Expressing